totalsiron as long as no agent capable of reducing iron(II1) is present. Reaction Mechanism. Absorption spectra were taken of various combinations of aluminum, iron, and PBBR with water as the blank (Figure 4). The two iron-containing samples absorb a t 400 mp, whereas PBBR and aluminum-PBBR absorb only dightly a t this wavelength. These measurements lead to the conclusion that iron destroys the aluminum-PBBR complex to form a stronger nonfluorescent complex with PBBR. Use of Superchrome Violet B. Weissler and White, in their method for aluminum, reported a greater interference from iron when Super-
chrome Violet B was used in place of PBBR. Consequently, a 0.1% solution of Superchrome Violet B in 9570 ethyl alcohol was prepared, and a standard curve for iron was determined. However, the sensitivity of the reagent was equal to the sensitivity obtained with PBBR. but the interference from cobalt was greater (Table 11), and therefore no further work was done along these lines. Stability. The fluorescent intensities were stable for a period of a t least 48 hours. Accuracy of the Method. NBS samples of nickel-silver (157a) reported to contain 0.174Oj, Fe were analyzed by the present method.
Copper and nickel, the major constituents in addition t o zinc, were first removed by electrolysis and precipitation as the dimethylglyoximate, respectively. The results obtained on two aliquots mere 0.176 and 0.171% Fe. LITERATURE CITED
(1) Diehl, H., Smith, G. F., “The Iron
Reagents.” G. F. Smith Chemical Co., Columbus, Ohio, 1960. (2) ~7eissler,A., White, C. E.. ANAL. CHEM.18, 530 (1946). RECEIVEDfor review June 22, 1962. Accepted August 31, 1962. Division of Analytical Chemistry, 142nd Meeting, ACS, Atlantic City, N. J., September 1962.
Semimicro and Micro Steam Distillation The Estimation of the Essential Oil Content of Small Plant Samples W. J. FRANKLIN and H. KEYZER Museum o f Applied Arts and Sciences, Sydney, New Soufh Wales, Australia
b Two micro steam distillation units have been designed to estimate the volatile oil content of small plant material samples. The results obtained are of an accuracy comparable to that of conventional macromethods, although the volumes of oil recovered amount to only a few microliters. In some instances a single leaf may constitute a sufficient sample. A semimicro steam distillation apparatus has also been developed for the same purpose. In addition, the three stills as a group can at least partially obviate the problems often encountered in the purification of small amounts of certain organic liquids.
T
HE MARKED INCREASE in the use of infrared spectroscopy and gas liquid chromatography in phytochemistry during the last decade has necessitated the development of suitable techniques for handling very small quantities of essential oil constituents. Furthermore, current botanical research into the genetics, nutritional requirements, and biosynthetic mechanisms of essential oil bearing plants necessitates the chemical investigation of very small amounts of plant tissue, involving the quantitative recovery of oil volumes ranging from 1 to 100 d. EXPERIMENTAL
Microdistillation units have previously been described ( 2 ) ; so also has a semimicro Apparatus and Procedure.
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ANALYTICAL CHEMISTRY
steam distillation assembly (2). However, none of these is suited to the needs described above. The specific need for a steam distillation technique arises from the known stability of some very reactive essential oil components to steam and the effective liberation of oil from the sacs in the plant tissue. Still 1. The apparatus show? in Figure 1 is termed a semimicrocohobation .till. The term “cohobation” implies circulative steam distillation, in which the distillate is condensed and allowed to separate into oil and water layers. the aqueous phase then being returned to the distillation vessel for further use. Still 1 is a modification of the IIcKern and Smith-White apparatiis (SI, but allows determination of oil quantities to a lower limit of 50 pl. The semimicroapparatus differs radically in the system of measurement. The leaf sample is weighed into a 250ml. flask containing sufficient water. The contents of the flask are boiled. The condensate enters C, and its momentum forces the oil into D, where the oil separates as the top layer while the water returns through the rubber tube F to the flask. On a 30-gram leaf sample, distillation is complete in 2.5 hours. A rubber stopper is inserted in C, the rubber tube F is clamped off, and stopcock E is closed. A hypodermic needle fixed to a separatory funnel G filled with saturated sodium chloride solution is inserted into the rubber tube F. Another needle, fitted to a graduated pipet I (size appropriate to the collected oil) is also inserted into F . On opening stopcock H , the pipet fills
with salt solution. Stopcock E is opened and the oil level in D is allowed to rise into the capillary to a convenient mark-for example, K-whereupon E and H are closed. The pipet is read and stopcock E is reopened. The oil is allowed to pass K until the bottom level reaches I
